Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 18 (1), 122

Synergistic Effect of Phytochemicals on Cholesterol Metabolism and Lipid Accumulation in HepG2 Cells

Affiliations

Synergistic Effect of Phytochemicals on Cholesterol Metabolism and Lipid Accumulation in HepG2 Cells

Ennian Leng et al. BMC Complement Altern Med.

Abstract

Background: Crocin (CRO), chlorogenic acid (CGA), geniposide (GEN), and quercetin (QUE) are all natural compounds with anti-obesity properties, in particular, hypolipidemic effects, which have been widely used for the treatment of obesity-related metabolic diseases. However, it is not yet known whether these compounds interact synergistically. Here, we investigated the effects and molecular mechanisms of CRO, CGA, GEN, QUE, and a combination of all four compounds (CCGQ), on lipid accumulation in human hepatoma (HepG2 cells).

Methods: The optimal concentration of CRO, CGA, GEN, QUE to stimulate HepG2 cells proliferation was determined using MTT assay. HepG2 cells were pretreated with 10 μmol/L simvastatin, 1 μmol/L CRO, 30 μmol/L CGA, 10 μmol/L GEN, 10 μmol/L QUE, and CCGQ (a combination of 1 μmol/L CRO, 30 μmol/L CGA, 10 μmol/L GEN, and 10 μmol/L QUE) for 24 or 48 h. Oil red O staining and extracellular TC and TG levels were detected. The RT-PCR was used to observe on cholesterol metabolism-related gene expression. Immunocytochemistry and western-blot assayed the 3-hydroxy-3-methylglutaryl-coenzyme (HMGCR) protein expression in HepG2 cells.

Results: Compared to those of control, we demonstrated that treating HepG2 cells for 48 h with CCGQ resulted in a strong synergistic effect, causing a marked decrease in lipid deposition in comparison to individual treatments, in both triglyceride and total cholesterol (CRO, 5.74- and 1.49-folds; CGA, 3.38- and 1.12-folds; GEN, 4.04- and 1.44-folds; QUE, 3.36- and 1.24-folds; simvastatin, 5.49- and 1.83-folds; and CCGQ, 7.75- and 2.20-folds), and Oil red O staining assays. In addition, CCGQ treatment increased ATP-binding cassette transporter (ABCA1), cholesterol 7α-hydroxylase (CYP7A1), and AMP-activated protein kinase 2α (AMPKα2) mRNA expression, while decreasing sterol regulatory element binding protein 2 (SREBP2), and liver X receptor alpha (LXRα) mRNA expression. Notably, CCGQ was more effective in decreasing HMGCR expression than the individual treatments.

Conclusion: The CCGQ combination has potential, both as a complementary therapy for hyperlipemia, and in preventing further obesity-related complications.

Keywords: Chlorogenic acid; Crocin; Geniposide; Lipid accumulation; Quercetin; Synergistic interaction.

Conflict of interest statement

Ethics approval and consent to participate

Not Applicable.

Consent for publication

Not Applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Chemical structures of CRO, CGA, GEN and QUE
Fig. 2
Fig. 2
The effects of different concentrations of CRO, CGA, GEN and QUE on the viability of HepG2 cells for 24 h or 48 h. Data are expressed as mean ± SEM from at least five independent experiments
Fig. 3
Fig. 3
The effects of CRO, CGA, GEN, QUE and C + C + G + Q on lipid content in HepG2 cells. HepG2 cells were treated with Oleate (0.1 mmol/L), SIM (10 μmol/L), CRO (1 μmol/L), CGA (30 μmol/L), GEN (10 μmol/L), QUE (10 μmol/L) and C + C + G + Q (1 μmol/L + 30 μmol/L + 10 μmol/L + 10 μmol/L) for 24 h or 48 h. a After 48 h incubation with different drugs, the images of cells were observed by microscope at 250 × original magnification showing lipid accumulation in cells stained by Oil red O. b The comparison of integral optical density (IOD) for Oil red staining in cells. Effects of different drugs on the secretion of (c) TC and (d) TG were observed by HepG2 cells in minimum essential medium. Data are expressed as mean ± SEM from at least four independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. Oleate. After 24 h, aP < 0.05 vs. control, bP < 0.05 vs. SIM. After 48 h, cP < 0.05 vs. control, dP < 0.05 vs. SIM
Fig. 4
Fig. 4
mRNA expression levels of the regulation cholesterol activities associated genes ABCA1, SREBP2, CYP7A1, LXRα, AMPKα2 and HMGCR in HepG2 cells. All genes were normalized with GAPDH. HepG2 cells were treated with (1) SIM (10 μmol/L), (2) C + C + G + Q (1 μmol/L + 30 μmol/L + 10 μmol/L + 10 μmol/L) (3) CRO (1 μmol/L), (4) CGA (30 μmol/L), (5) GEN (10 μmol/L), (6) QUE (10 μmol/L) and no drug for (7) Control respectively for 48 h. Data represent as mean ± SEM of three independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. SIM
Fig. 5
Fig. 5
Expression of HMGCR in HepG2 cells. a Representative immunocytochemical images of HMGCR expression in cells. HepG2 cells were treated with SIM (10 μmol/L), C + C + G + Q (1 μmol/L + 30 μmol/L + 10 μmol/L + 10 μmol/L), CRO (1 μmol/L), CGA (30 μmol/L), GEN (10 μmol/L) and QUE (10 μmol/L) for 48 h respectively, and treated with PBS instead of the primary antibody of HMGCR for negative control, and with no drug for normal control. The brown granules in the cytoplasm represent HMGCR positive staining results. b The bar chart showing the comparison of the IOD for HMGCR expression in cells. Data represent as mean ± SEM of three independent experiments. *P < 0.05 vs. control, #P < 0.05 vs. SIM
Fig. 6
Fig. 6
All drugs and their combination suppress HMGCR protein expression in HepG2 cells. a Western blotting analysis of HMGCR, (b) The densitonmetric scanning of HMGCR after normalization with GAPDH. HepG2 cells from 3 set of each group were pooled separately and homogenates was prepared for western blotting analysis of HMGCR. HepG2 cells were treated with QUE (10 μmol/L), CRO (1 μmol/L), CGA (30 μmol/L), GEN (10 μmol/L), SIM (10 μmol/L) and C + C + G + Q (1 μmol/L + 30 μmol/L + 10 μmol/L + 10 μmol/L) for 48 h respectively. Data represent as mean ± SEM of three independent experiments. *P < 0.05 vs. control

Similar articles

See all similar articles

Cited by 3 articles

References

    1. Bener A, Yousafzai MT, Darwish S, Al-Hamaq AO, Nasralla EA, Abdul-Ghani M. Obesity index that better predict metabolic syndrome: body mass index, waist circumference, waist hip ratio, or waist height ratio. J Obes. 2013;2013:269038. doi: 10.1155/2013/269038. - DOI - PMC - PubMed
    1. Stefan N, Haring HU, Hu FB, Schulze MB. Metabolically healthy obesity: epidemiology, mechanisms, and clinical implications. Lancet Diabetes Endocrinol. 2013;1:152–162. doi: 10.1016/S2213-8587(13)70062-7. - DOI - PubMed
    1. Walther TC, Farese RV., Jr Lipid droplets and cellular lipid metabolism. Annu Rev Biochem. 2012;81:687–714. doi: 10.1146/annurev-biochem-061009-102430. - DOI - PMC - PubMed
    1. Lodhi IJ, Yin L, Jensen-Urstad AP, Funai K, Coleman T, Baird JH, et al. Inhibiting adipose tissue lipogenesis reprograms thermogenesis and PPARgamma activation to decrease diet-induced obesity. Cell Metab. 2012;16:189–201. doi: 10.1016/j.cmet.2012.06.013. - DOI - PMC - PubMed
    1. Ramasamy I. Update on the molecular biology of dyslipidemias. Clin Chim Acta. 2016;454:143–185. doi: 10.1016/j.cca.2015.10.033. - DOI - PubMed
Feedback